It is well established that exposure to environmental stressors or toxicants such as endocrine-disrupting chemicals (EDCs) during in utero and/or neonatal development can result in adult-onset diseases. Such detrimental changes have been linked to aberrant DNA methylation patterns as well as other epigenetic mechanisms. It is of great concern that recent studies have suggested that such disease susceptibilities can be transmitted via epigenetic mechanisms to subsequent generations that were never directly exposed to the EDCs. For example, testicular abnormalities were transmitted to F2-F4 generation males via the male germ line after in utero exposure to an EDC. However, these effects were found to be sex-specific since F2-F3 females exhibited milder phenotypes that required exposure of both parents to the EDC. In this proposal, our objective is to address the critical issue of whether the female germ line is involved in such transgenerational (TG) effects of EDC exposure. Methoxychlor (MXC) is an EDC that has actions mimicking those of many other EDCs in the environment and was shown to cause epigenetic changes in the gonad; it will therefore be used as a model EDC in this proposal. Our hypothesis is that exposure to MXC during the critical window of development, defined here as the period of gonad differentiation through oocyte epigenetic reprogramming, leads to TG effects in the ovary that are mediated by epigenetic changes in the oocyte. To test our hypothesis, the proposal has two interrelated but distinct specific aims: (1) Determine somatic TG epigenetic effects of MXC in the ovary of F3 generation following exposure of F1. F1-generation inbred female rats will be treated with 0, 20 5g/kg/day MXC, or 50-100 mg/kg/day MXC between E11 and PND7. These females will be bred with untreated males, and F2-F4 females will be generated. Initially, ovarian morphology and gene expression and fertility will be assessed only in the F3 females since observation of TG effects in F3 generation is the minimum requirement for establishing TG effects. Gene expression changes and global and gene-specific DNA methylation changes will be investigated using conventional methods as well as array-based approaches. (2) Investigate the epigenetic changes induced by MXC in F1/F2 oocytes that may lead to TG effects in F3. DNA methylation patterns in imprinted genes and parasitic DNA sequences such as retrotransposons in oocytes will be analyzed, as aberrant methylation patterns in these sequences can be pathogenic. Oocytic gene expression patterns will also be examined; any correlation between the changes in gene expression and DNA methylation will be elucidated. The proposed study will be the first to investigate the epigenetic TG effects of EDCs transmitted via the female germ line beyond the F2 generation. We recognize the considerable risk associated with this study, but implications to the health of present and future generations are significant given the widespread presence of EDCs in the environment. A mechanistic understanding of the TG epigenetic actions of MXC will broaden our knowledge of how EDCs, in general, disrupt normal epigenetic programming in females, and facilitate the development of strategies to prevent and treat EDC-induced TG abnormalities.